The invention relates to compositions and methods for the diagnosis and treatment of Huntington""s disease. More specifically, the invention provides a cell line that stably expresses truncated mutant huntingtin protein for use as a model for Huntington""s disease, antigens and monoclonal antibodies to mutant huntingtin protein, and methods for their use.
Huntington""s disease (HD) is a progressive neurodegenerative disorder characterized by motor disturbance, cognitive loss and psychiatric manifestations (Martin and Gusella (1986), N. Engl. J. Med. 315:1267-1276. It is inherited in an autosomal dominant fashion, and affects about 1/10,000 individuals in most populations of European origin (Harper, P. S. et al., in Huntington""s Disease, W. B. Saunders, Philadelphia, 1991). The hallmark of HD is a distinctive choreic movement disorder that typically has a subtle, insidious onset in the fourth to fifth decade of life and gradually worsens over a course of 10 to 20 years until death. Occasionally, HD is expressed in juveniles typically manifesting with more severe symptoms including rigidity and a more rapid course. Juvenile onset of HD is associated with a preponderance of paternal transmission of the disease allele. The neuropathology of HD also displays a distinctive pattern, with selective loss of neurons that is most severe in the caudate and putamen regions of the brain. The biochemical basis for neuronal death in HD has not yet been fully explained, and there is consequently no treatment effective in delaying or preventing the onset and progression of this devastating disorder.
The molecular basis of HD is the expansion of a CAG repeat encoding a polyglutamine tract in the N-terminus of the HD protein huntingtin (Huntington""s Disease Collaborative Research Group (1993) Cell 72:971-983). The length of the polyglutamine repeat is inversely correlated with the age of disease onset. A breakthrough in HD research was the development of transgenic mouse models for the disease. Transgenic mice expressing exon-1 of the human HD gene with an expanded CAG repeat develop a progressive neurological disorder that exhibits many of the features of HD, including choreiform-like movements, involuntary stereotypic movements, tremor, and epileptic seizures (Mangiarini et al. 1996 Cell 87:493-506). These mice have CAG repeats lengths of 141-157 while the huntingtin protein of normal mice contains 35 or fewer CAG repeats. Expansion of a CAG or glutamine repeat is associated with seven other inherited neurological disorders (MacDonald et al. (1996) Curr. Opin. Neurobiol. 6:638-643; Reddy and et al. (1997) Curr. Opin. Cell. Biol. 9:364-372; Ross (1997) Neuron 19:1147-1150).
Normally, huntingtin is a cytoplasmic protein characterized by high levels of expression in the striatal neurons vulnerable to degeneration in HD and low or undetectable expression levels in neurons resistant to degeneration (Ferrante et al. (1997), Science 227:770-773). Increasing evidence has shown that the expansion of the glutamine repeat in the huntingtin protein causes small protein fragments to accumulate and aggregate in the nucleus of cells. For instance, transgenic mice (Bates mice) expressing exon-1 of the HD gene containing  greater than 115 CAGs have neuronal intranuclear inclusions before they develop neurological disorders (Davies et al., (1997) Cell 90:537-548). Moreover, intranuclear aggregates containing N-terminal huntingtin fragments have been observed in the brains of HD patients (DiFiglia et al., (1997) Science 277:1990-1993); Becher et al., (1998) Neurobiol. Dis. 4:387-397; Gutekunst et al., (1999) J. Neurosci. 19:2522-2534).
The association between HD and nuclear aggregates has led to the hypothesis that such nuclear aggregates arc toxic and play a causative role in the pathology of HD. In fact, several studies using cultured cells have shown that nuclear aggregates of polyglutamine proteins are associated with cell death (Cooper et al. (1998) Hum. Mol. Genet. 7:783-790); Martindale et al. (1998) Nat. Genet. 18:150-154; Hackam et al. (1999) Hum. Mol. Genet. 8:25-33; Moulder et al. (1999) J. Neurosci. 19:705-715). However, other studies show that the nuclear localization of polyglutamine proteins, not the formation of aggregates, is critical for neuronal pathology in transgenic mice (Klement et al. (1998) Cell 95:41-53) and in cultured striatal neurons (Saudou et al. (1998) Cell 95:55-66). In addition, the regional distribution of intranuclear aggregates in HD brains does not correspond to the neuropathology (DiFiglia et al., 1997; Becher et al., 1998; Gutekunst et al., 1999).
Despite the controversy surrounding the role of huntingtin aggregates in the pathology of HD, it is clear that expanded polyglutamine causes huntingtin to accumulate in the nucleus. Because many transcription factors contain a glutamine-rich domain and this domain can regulate their activity (Courey et al. (1988) Cell 55:887-898; Courey et al. (1989) Cell 59:827-836 ; Gerber et al. (1994) Science 263:808-811), it has been proposed that expanded polyglutamine-containing proteins interfere with gene transcription when they are localized to the nucleus. This possibility also provides a common mechanism to explain the features that HD shares with other glutamine-repeat diseases, including spinobulbar muscular atrophy and the spinocerebellar ataxias.
A HD cell model would be a useful tool in elucidating the mechanistic basis of HD. Most of the reported cell models have used transient transfection in which the expression levels of transfected protein vary greatly and influence aggregation of the transfected protein and cell viability. A stably transfected cell line that expresses mutant huntingtin is needed to provide a suitable approach to study whether intranuclear huntingtin affects cellular function at the transcription level, as well as other questions concerning the molecular basis of HD. Such a cell line would also allow for the establishment of a biochemical assay yielding consistent and reproducible results, which would greatly facilitate the screening and evaluation of potential HD therapeutic agents.
The present invention provides methods and compositions for the study, diagnosis, and treatment of Huntington""s disease. Compositions comprise stably transfected cell lines, an antigen associated with Huntington""s disease, and monoclonal antibodies specific for said antigen. The invention provides neuronal cell lines for use as a cellular model of Huntington""s disease. More specifically, the invention provides rat pheochromocytoma PC12 cell lines that stably express truncated mutant huntingtin protein containing an expanded polyglutamine repeat. By xe2x80x9cexpressxe2x80x9d is intended that the cell line synthesizes the truncated mutant huntingtin protein, including the steps of transcribing, translating, and assembling truncated mutant huntingtin. These cell lines display the cellular defects observed in cells of patients with Huntington""s disease. The PC12 cell lines of the invention can be maintained in culture for an extended period as continuous stable cell lines. The cells retain the morphological characteristics of the primary cells from which they were derived and the phenotypic traits associated with cells derived from animal models of Huntington""s disease and Huntington""s disease patients.
The methods and compositions of the invention find use in the study of the pathological mechanism of the mutant huntingtin in Huntington""s disease and the development of therapeutic strategies for this disease.
In another embodiment of the invention, the methods and compositions are used in a screening assay to discover and evaluate therapeutic molecules for the treatment of Huntington""s disease. The PC12 cell lines of the invention provide predictive in vitro test results of the effectiveness of therapeutic molecules, and can be used in a high through-out drug screening assay prior to screening of potential therapeutic molecules using animal models of the disease. This cell model reduces the cost and increases the efficiency of screening for such therapeutic molecules.
In another embodiment, monoclonal antibodies of the invention are used in a diagnostic assay for Huntington""s disease. By xe2x80x9cdiagnostic assayxe2x80x9d is intended an assay designed to distinguish one disease, in this case Huntington""s disease, from other diseases with a similar clinical presentation. The monoclonal antibodies of the invention are without cross-reactivity to other proteins containing polyglutamine repeats, and react strongly with mutant huntingtin but weakly with wild-type rodent huntingtin. The diagnostic assay for Huntington""s disease of the invention uses pre- or post-mortem tissue from an individual as a sample, and utilizes the Huntington""s disease antigen and antibodies of the invention. The samples from the patient include, in addition to brain tissue, other tissues and cells including skeletal muscle, skin, heart, liver, adrenal glands, pancreas, kidney, stomach wall, duodenum, and mucosal cells.